Alpha-lactalbumin (alpha-LA)-like proteins modify the activity of N-acetylglucosa-minide beta 1-4 galactosyltransferase. Mammary gland alpha-LA bears sequence homology with lysozyme. In mammary gland alpha-LA gene expression is regulated by insulin, hydrocortisone, prolactin and progesterone. Previously we have sequenced mammary alpha-lactalbumin gene and shown that: a) 5'-flanking sequence has several short repeat sequences and some of which are similar to the concensus sequence for the binding site of hydrocortisone and progesterone receptor; b) the functional domain of alpha-LA which contains amino acids essential for its interaction with galactosyltransferase, and is conformationally different from the corresponding region of lysozyme, is coded by a separate nucleotide region preceded by two (TG)n repeats. In the present studies the regulatory elements of alpha-LA gene are being localized. Various regions of alpha-LA gene have been introduced into chloramphenicol (CAT)-vectors and tested in mammalian transfection experiments for their ability to influence CAT-expression. Alpha-LA gene sequences which become DNAase hypersensitive and bind specific proteins during different stages of mammary gland development have been studied. The results show: (a) 700 bp DNA fragments from the 3'-end of alpha-LA gene, which contains most of the third intervening sequence with its two (TG)n repeats - (TG)21 and (TG)24 - and a part of the 4th exon which codes for the functionally important region of alpha-LA, acts in an enhancer fashion in CAT-assays; (b) activity of this enhancer is cell type specific; (c) 5'-flanking sequences of alpha-LA gene show DNAase hypersensitive regions during different stages of mammary gland differentiation; (d) nuclear protein fractions extracted from mammary gland nuclei bind to specific regions of 5'-flanking sequence of alpha-LA gene.